MATERIALS AND METHODS
Reagents
DEPC water or ultrapure water, Trizol and miRNA probes were purchased from Life
Technologies. dNTP, AMV reverse transcription buffer, and AMV reverse transcriptase were
purchased from three different companies (Companies 1-3). Taq polymerase and buffer were
purchased from Life Technologies. All other reagents were of analytical grade.
Human serum samples and experimental chicks
All human blood samples were collected from healthy patients at the Jinling Hospital
(Nanjing, China) at the time of physical examination. All of the donors or their guardians
provided written consent, and this study was approved by the Hospital Ethical Examination
Committee of Human Research of Nanjing Jinling Hospital, (Nanjing, China) and written
informed consent was obtained from each participant. Whole blood was separated into serum
and cellular fractions within 2 h after the blood was collected.
Plasma samples were taken from 3 chicks brought from Qianyuan Hao Bio-pharmaceutical
companies (Nanjing, China). Seven-day-old chicks were killed, and their plasma was
collected. The birds were sacrificed under the NIH Guidelines for the Care and Use of
Laboratory Animals, and the animal experiment protocol was approved by the Animal Care
Committee of Nanjing University (Nanjing, China).
RNA isolation and RT-qPCR
One hundred microliters of human serum was diluted with 300 l of DEPC water, and the
total RNA was extracted with Trizol reagent (Life Technologies, Carlsbad, CA, USA)
according to the manufacturer’s instructions. To extract the RNA from commercial reverse
transcriptase, 10 l or 160 l of AMV solution was diluted with three volumes of DEPC water,
and the total RNA was extracted with Trizol reagent. Chick blood was drawn by heart
puncture (3~5 ml per chick) into 10 ml negative pressure blood collection tubes containing
EDTA potassium salt. Blood cells were sedimented by centrifugation at 3000 g for 10 min,
and the upper plasma level was collected. Total RNA was isolated with Trizol reagent
(Invitrogen) according to the manufacturer’s instructions. Glycogen (Invitrogen) was added to
the RNA precipitation step. The total RNA samples were dissolved in ultrapure water (Life
Technologies, Carlsbad, CA, USA). To perform the reverse transcription reaction, 1 g of
total RNA from serum samples or the same volume of ultrapure water (no-template-control)
was added to the 10 l reverse transcription reaction systems (using commercial company
products company 1-3). In some experiments, 3 l of total RNA was extracted and reverse
transcriptase was added to the 10 l reverse transcription reaction systems. A reverse
transcription system (TaqMan MicroRNA Reverse Transcription Kit, Applied Biosystems,
USA) was used in this study, and it employs a recombinant Moloney murine leukemia virus
(rMoMuLV) reverse transcriptase called “MultiScribe Reverse Transcriptase”. This
rMoMuLV system can provide a standard reverse transcriptase that is free of miRNA.
Real-time PCR was performed with a TaqMan PCR kit and an Applied Biosystem 7500
Sequence Detection System (Applied Biosystem). All reactions were performed in triplicate,
and no-template controls were included. After the reaction, the CT values were determined
using fixed threshold settings.
Solexa sequencing
The Solexa sequencing was performed by BGI Tech company (China, Shenzhen). Briefly,
after the PAGE purification of small RNA molecules and the ligation of a pair of Solexa
adaptors to the 5’ and 3’ ends, a 90 base pair fragment (small RNA + adaptor) was amplified
by using the adaptor primers for 17 cycles and then isolated on PAGE gels. The isolated DNA
sequence was then used for the cluster generation and sequencing analysis using an Illumina
Genome Analyzer according to the manufacturer’s instructions. Digital data resulted from the
image files generated by the sequencer; a series of procedures were subsequently performed
including summarizing data production, evaluating sequencing quality and depth, calculating
the length distribution of small RNAs, and filtering out contaminated reads. The clean reads
were aligned against the miRBase 16.0 database based on the Smith-Waterman algorithm
after constructing the adaptor sequences. Matching miRNAs were counted only after
identifying the sequence and length in comparison to reference miRNAs.
Ultrafiltration
Amicon Ultra-0.5 Centrifugal Filter Devices (Millipore, 10K) were used to filter the AMV
solution. Briefly, 40 l of AMV solution was diluted to 500 l with 1 × reaction buffer and
added into the Amicon Ultra-0.5 filtration tube (10 kDa). After centrifugation at 14,000 g for
15 minutes, approximately 40 l of the remaining solution above the filter membrane was
collected immediately according to the manufacturer’s instructions. To test the reverse
transcription function of filtered AMV, a small aliquot of the samples was used in the
RT-qPCR system as the reverse transcriptase (synthetic miR-16 RNA molecules served as the
RNA template). The remaining solutions were extracted with Trizol to extract the RNA
contents from the AMV. The extracted RNAs were quantified by MMLV-based RT-qPCR. An
equal volume of unfiltered AMV was used as a control AMV to perform the miR-16 reverse
transcription or RNA extraction and quantification.
Statistical analysis
All the RT-qPCR data points are representative of at least three independent experiments and
are presented as the means ± SEM. Prism 5.0 software (GraphPad, Inc.) was used for the data
analysis.